ABSTRACT Entamoeba histolytica causes dysentery and liver abscesses with invasive disease in 50 million people annually. Despite being an important human pathogen, this organism is poorly studied, has very limited therapeutic options, and has no significant drug development pipeline. Thus, there is a critical need for new drugs against this pathogenic ameba. We chose a compound screening approach using the ReFRAME library, which was generated by Calibr and the Bill and Melinda Gates Foundation to have high-value hits with the vast majority of compounds having been tested in humans. We have completed screening of ~11,000 compounds against E. histolytica and identified 156 primary (~1.3%) hits. We subsequently performed confirmatory testing and confirmed 50 hits with EC50 15M. By considering FDA approval status, clinical trial data, hepG2 cell toxicity and potency against E. histolytica trophozoites, we identified 19 high-priority compounds for further testing in this grant cycle. Of these 19 priority compounds, 9 have been FDA approved or have FDA orphan designation and an additional 7 have been in late stage human clinical trials (Phase II or III); 14 have improved efficacy compared to metronidazole. Thus, these 19 compounds are great options for a repurposing approach. In this R21 grant, we will characterize these high value hits in the following two aims. In Aim 1, we will test these compounds against Entamoeba cysts (encysting cells and mature cysts), metronidazole resistant Entamoeba, and clinical isolates. We will also determine the kinetics of parasite killing with each compound and the delay of parasite recrudescence after drug removal. Based on these results and analysis of pharmacokinetic data on the compounds, in Aim 2, we will select ~2-5 select compounds to test in vivo efficacy in a mouse model of Entamoeba colitis. Successful completion of this work will identify ~2 lead compounds/series with activity against Entamoeba histolytica. Based on our experience with amebae, significant drug development expertise at Stanford and UCSD, and a cadre of outstanding collaborators, we are confident of a successful outcome.